The mean cell survival was calculated from three independent experiments. Sensitivity to metronidazole was also

evaluated using E-test strips on BHISA according to the manufacturer’s instructions (AB Biodisk, Solna, Sweden). Bacteroides fragilis 638R and recQ mutant cells were grown on BHISA before colonies were scraped off and resuspended in phosphate-buffered saline (PBS). Aliquots of these suspensions were fixed in glutaraldehyde (2% v/v in PBS) and prepared for transmission electron microscopy (TEM) (Simpson et al., 2006). Ultrathin sections were viewed using a JEOL 1200EX II TEM. Further cell samples were either Gram stained or the DNA ABT-263 datasheet and membranes were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg mL−1) and FM4-64 (1 mM), respectively. Fluorescence microscopy was performed at × 1000 magnification using a Zeiss Axiovert 200 microscope and photographed using a Zeiss Axiocam. Pictures were analysed using axiovision 4.6. To visualize DNA strand breaks, genomic DNA was extracted and the presence of double- and single-strand breaks was analysed by neutral and alkaline agarose gel electrophoresis, respectively (Abratt et al., 1990; Dachs et al., 1995). Analysis of the B. fragilis 638R genome sequence revealed the presence of three putative RecQ genes, identified by ORF numbers BF638R_3282 (Q1), BF638R_3781 (Q2) and BF638R_3932 (Q3). These ORFs encoded deduced proteins of 607

amino acids (aa), OSBPL9 634 aa and 726 aa in length, respectively. These PF-01367338 cell line B. fragilis 638R loci corresponded to BF3249, BF3706 and BF3892, respectively, from strain NCTC 9343. Q1 was most similar to E. coli RecQ (43.7% aa identity), while Q2 and Q3 showed 39.6% and 38.9% aa identity to it, respectively. The presence of multiple RecQ homologues in prokaryotes had not been described before this study on B. fragilis,

although it is well documented in higher eukaryotes. For example, the human genome encodes five RecQ proteins (BLM, WRN, RecQL1, RecQL4, RecQL5), while Arabidopsis thaliana encodes seven RecQ homologues (Hartung & Puchta, 2006). Our analysis revealed that the annotated genomes from 14 members of the genus Bacteroides encoded multiple putative RecQ homologues (Fig. 1a; Table S2). The significance of multiple RecQ homologues in Bacteroides is unclear. All the B. fragilis 638R RecQ homologues contained two of the signature amino acid domains representative of all known RecQ helicases, namely a helicase domain (with the essential DEXX motif [X representing alanine (A), histidine (H), serine (S) or aspartate (D) residues]) as well as a helicase C-terminal domain (Fig. 1b). The helicase domain from all three homologues further contained the conserved regions 0 to VI (Bennett & Keck, 2004). The BF638R_3932 (Q3) homologue contained an incomplete HRDC domain, whereas the RecQ coded by BF638R_3781 (Q2) was unusual as it completely lacked an HRDC domain.

, 2008). For each of the 84 genes, PCR analyses confirmed the location of the transposon and demonstrated the absence of an intact copy of the gene. The

321 genes GSK3235025 inactivated in the original library and the 84 additional genes inactivated in the minitransposon library bring the total number of inactivated genes in M. pulmonis to 405. None of the genes coding for RNA species were disrupted in the transposon libraries. The 1.4-kb NADH oxidase gene (MYPU_0230) was disrupted in the minitransposon library. In the original library, transposon insertions mapped to this gene in 27 transformants, but in each case, additional PCR analyses failed to confirm the position of the transposon in MYPU_0230 (French et al., 2008). Because the minitransposon inactivated genes thought to be essential, such as MYPU_0230, the distribution of the transposon insertion sites was examined for both libraries. The distribution was selleck products found to be highly similar (Fig. 1). Most of the differences may be due to random chance, with the exception of two hot spots for transposon insertion that were identified in the original library as HS1 and HS2 (French et al., 2008). In the minitransposon library, the density of transposon insertion sites within HS1 and HS2 was not higher than that for other regions and

hence the distribution of transposon insertions may be more uniform. Because there were no substantial differences in the distribution of transposon insertion sites in the libraries, alternative explanations for the inactivation of what were previously thought to be essential genes were considered. One possibility was that some nonessential genes are required for optimal growth and mutants with these genes disrupted were lost from the original library due to transposon excision, which is known to occur precisely at a high frequency (Mahairas et al., 1989; Krause

et al., 1997). Growth curves were performed and the doubling times were calculated as described Sclareol (Dybvig et al., 1989). The wild-type parent and a transformant that contained the minitransposon at an intergenic site had doubling times of 2.0 h, with an SD of 0.1 h. The minitransposon mutant with a disruption in the NADH oxidase gene had a doubling time of 3.2 h (SD=0.1 h). With a reduction in growth rate by 50%, ample opportunity exists for revertants to eventually dominate a culture. Tn4001 excision is often precise (Mahairas et al., 1989) and occurs at a high frequency in M. pulmonis (Dybvig et al., 2000). Thus, reversion due to loss of the transposon would be commonplace when using Tn4001T but not when using the minitransposon. Orthologs of 18 of the 84 genes knocked out in the minitransposon library but not in the original library were identified previously (Glass et al., 2006) as being essential in M. genitalium (Table 1). These 18 genes lack any obvious paralog in M. pulmonis that might have compensated for the gene loss. Many of these 18 genes may be similarly nonessential in M.

2c). No cleavage was observed when the XerSY314F mutant was used instead of the wild-type protein (data not shown). The vector pBEA756 possesses both gram-positive thermosensitive (Ts) and ColE1 replication origins. An internal fragment of the S. suis xerS gene was generated by PCR and cloned into this vector, generating the plasmid pBEA756XerCint. This plasmid was then successfully introduced into S. suis by electroporation

as described in section ‘Growth conditions and DNA manipulations’. At the restrictive temperature (37 °C), homologous recombination events were selected for by maintaining growth in the presence of kanamycin. selleck chemicals A single crossover event between the cloned xerS gene on the plasmid and the chromosomal copy of xerS resulted in the inactivation of the xerS gene, which was confirmed by PCR and by Southern blot (data not shown). Microscopic analysis of xerS mutant cells showed a significant increase in average chain length, with most of wild-type cells being 5–10 cells long, while mutants were more

than 10 cells long; in addition, extremely long chains, containing more than 30 cells, were also observed (Fig. 3). The re-introduction of a functional xerS with pGXerCF (pGhost9) restored the wild-type phenotype (data not shown). In this report, we described the purification and inactivation of the S. suis xerS gene and its MBP-fused product. The S. suis XerS recombinase was overexpressed and purified as a maltose-binding STK38 protein fusion, as previous work with XerCD recombinases has indicated that the N-terminal MBP moiety has no significant effect on Xer binding, cleavage buy BGJ398 or strand transfer activity (Blakely et al., 1997, 2000; Neilson et al., 1999; Villion & Szatmari,

2003). The difSL site was located about 50 bp before the start of the xerS coding region, as was found for most of the lactococci and streptococci (Le Bourgeois et al., 2007). In addition, XerS of S. suis displays 70% identity and 82% similarity to XerS of Lactococcus lactis (Le Bourgeois et al., 2007). Specific binding of difSL was detected at MBP-XerS concentrations of 3.43 nM and above, in the presence of a 1000-fold molar excess of poly dIdC competitor (Fig. 1a). The observation of more than one complex suggests that MBP-XerS is binding to both half-sites of difSL, which is consistent with other systems using one recombinase like Flp and Cre. Binding to the left half-site was detected, while virtually no retarded bands were visible in reactions on the right half-site (Fig. 1b,c), in agreement with results found by Nolivos et al. (2010) on the lactococcal difSL site. The faster migrating bands correspond to the binding of a single XerS monomer on the DNA, while the slower migrating forms correspond to the binding of two or more XerS protomers on the DNA. The additional retarded complexes seen with the difSL left half-site are most likely additional monomers binding to the complex via protein–protein interactions.

2c). No cleavage was observed when the XerSY314F mutant was used instead of the wild-type protein (data not shown). The vector pBEA756 possesses both gram-positive thermosensitive (Ts) and ColE1 replication origins. An internal fragment of the S. suis xerS gene was generated by PCR and cloned into this vector, generating the plasmid pBEA756XerCint. This plasmid was then successfully introduced into S. suis by electroporation

as described in section ‘Growth conditions and DNA manipulations’. At the restrictive temperature (37 °C), homologous recombination events were selected for by maintaining growth in the presence of kanamycin. www.selleckchem.com/products/ganetespib-sta-9090.html A single crossover event between the cloned xerS gene on the plasmid and the chromosomal copy of xerS resulted in the inactivation of the xerS gene, which was confirmed by PCR and by Southern blot (data not shown). Microscopic analysis of xerS mutant cells showed a significant increase in average chain length, with most of wild-type cells being 5–10 cells long, while mutants were more

than 10 cells long; in addition, extremely long chains, containing more than 30 cells, were also observed (Fig. 3). The re-introduction of a functional xerS with pGXerCF (pGhost9) restored the wild-type phenotype (data not shown). In this report, we described the purification and inactivation of the S. suis xerS gene and its MBP-fused product. The S. suis XerS recombinase was overexpressed and purified as a maltose-binding Dichloromethane dehalogenase protein fusion, as previous work with XerCD recombinases has indicated that the N-terminal MBP moiety has no significant effect on Xer binding, cleavage GSK1120212 concentration or strand transfer activity (Blakely et al., 1997, 2000; Neilson et al., 1999; Villion & Szatmari,

2003). The difSL site was located about 50 bp before the start of the xerS coding region, as was found for most of the lactococci and streptococci (Le Bourgeois et al., 2007). In addition, XerS of S. suis displays 70% identity and 82% similarity to XerS of Lactococcus lactis (Le Bourgeois et al., 2007). Specific binding of difSL was detected at MBP-XerS concentrations of 3.43 nM and above, in the presence of a 1000-fold molar excess of poly dIdC competitor (Fig. 1a). The observation of more than one complex suggests that MBP-XerS is binding to both half-sites of difSL, which is consistent with other systems using one recombinase like Flp and Cre. Binding to the left half-site was detected, while virtually no retarded bands were visible in reactions on the right half-site (Fig. 1b,c), in agreement with results found by Nolivos et al. (2010) on the lactococcal difSL site. The faster migrating bands correspond to the binding of a single XerS monomer on the DNA, while the slower migrating forms correspond to the binding of two or more XerS protomers on the DNA. The additional retarded complexes seen with the difSL left half-site are most likely additional monomers binding to the complex via protein–protein interactions.

005) in the tenofovir DF arm. In both the stavudine arms, significant increases in anthropometric measures occurred at 24 weeks but these decreased

by week 48. Mitochondrial toxicities occurred in both the stavudine arms. Immunological and virological outcomes were similar for all three arms. This study highlights the occurrence of metabolic abnormalities with both stavudine and tenofovir DF treatment. Awareness of the potential increased cardiovascular risk should be of concern with the use of both these therapies. “
“In order to estimate HIV incidence among high-risk groups, in January 2009 the Health Protection Agency introduced the Recent Infection Testing Algorithm (RITA) in England and Northern Ireland (E&NI), currently the only regions to inform patients of RITA results. This survey of HIV specialists Selleckchem CX-4945 aimed to investigate

the role of RITA in patient management and explore clinicians’ views on its role in clinical practice and during partner notification. An online questionnaire was distributed check details to HIV specialists via the British HIV Association membership email list in February 2011. Forty-two HIV specialists from 32 HIV centres responded to the survey among 90 centres enrolled in the programme (response rate 36%). Testing for recent infection was considered standard of care by 83% of respondents, 80% PAK5 felt confident in interpreting results and 92% discussed results with patients,

particularly in the context of a possible HIV seroconversion illness (96%) or when deciding when to start antiretroviral therapy (70%). A third (36%) of specialists were initially concerned that RITA results may cause additional anxiety among patients; however, no adverse events were reported. The majority (90%) felt that results could assist with contact tracing by prioritizing patients with likely recent infection. However, only a few centres have currently incorporated RITA into their HIV partner notification protocols. RITA has been introduced into clinical practice with no reported patient adverse events. Access to results at centre level should be improved. National guidance regarding use of RITA as a tool for contact tracing is required. Large-scale use of Tests for Recent Infection (TRI) for HIV allows a better understanding of transmission dynamics of the HIV epidemic and an estimation of HIV incidence among high-risk groups [1]. The Health Protection Agency (HPA) in collaboration with HIV service providers introduced the Recent Infection Testing Algorithm (RITA) nationally in England and Northern Ireland (E&NI) in January 2009, although individual centres had previous experience with HIV incidence testing [2]. So far, over 4000 serum samples from 90 clinical centres have been tested for recent infection [3].

05. At the beginning and end of each electrode tract, two X-radiographs (coronal and sagittal planes)

were taken to identify the initial and final positions of the microelectrode tip in the brain. From these X-radiographs, the spatial locations of the electrode tip at the beginning and end of each electrode penetration could be accurately defined with respect to the posterior lip of the sphenoid bone – a bony promontorial landmark in the skull clearly visible in X-radiographs (Aggleton & Passingham, 1981). As a result, the location of the electrode tip with reference to the known defined laminar cytoarchitecture of mPFC could, to a first approximation, be assessed from a stereotaxic X-radiographic atlas of the macaque brain (Feigenbaum & Rolls, 1991) in conjunction with the standard laboratory atlas for macaques of Paxinos et al. (2000). (The positions of electrode tracts Maraviroc supplier were subsequently confirmed histologically in serial Nissl-stained sections through mPFC – see Fig. 1A.) Using the posterior lip of the sphenoid bone as reference, the positions of

each recorded cell along the path of each electrode tract could be accurately mapped in the coronal (mediolateral) and sagittal (anteroposterior) planes. By consulting monkey brain atlases (Aggleton & Passingham, 1981; Feigenbaum & Rolls, 1991; Paxinos et al., 2000) the areal locations of each recorded neuron could be defined Selleck Epacadostat reliably. At the end of all experimental

work, electrolytic microlesions were made through the tip of a recording electrode to mark the locations of typical neurons in the mPFC of each hemisphere for both BM and BN. The animals were deeply anaesthetized with sodium pentobarbitone (Sagatal) and transcardially perfused, initially with physiological saline (0.9%) and subsequently with 0.1 m phosphate-buffered (PB) 4% paraformaldehyde (pH 7.4 at room temperature). The brains remained in the skulls overnight before being carefully dissected from the cranium. Tryptophan synthase Following infiltration with graded sucrose solutions (10, 20 and 30%), complete sets of serial 1-in-2 sections (50 μm thick) from the entire rostrocaudal extent of each brain were then prepared in the coronal plane using a freezing microtome. Sections were collected into 0.1 m PB and subsequently mounted in order onto glass slides and air-dried. Finally, the sections were stained with cresyl violet to reveal areal and laminar cytoarchitectures then passed through an ascending series of alcohols before being embedded in DePeX mountant and coverslipped. The microlesions together with the associated X-radiographs and stereotaxic atlases enabled the areal positions of all cells to be reconstructed from the Nissl-stained sections using the method of Feigenbaum & Rolls (1991).

We thank Prof. F. Stewart (Technische Universität Dresden, Germany) for the provision of plasmids pR6K-rpsL-Neo and pSC101-BAD-Cre-tet and Dr T.D. Ho for the provision

of plasmid pKD46. H.N.T. is a recipient of IRO scholarships from Katholieke Universiteit Leuven, Belgium. “
“Per-ARNT-Sim (PAS) domains are important signalling modules that possibly monitor changes in various stimuli such as light. For the majority of PAS domains that have been identified by sequence similarity, the biological function of the signalling pathways has not yet been experimentally investigated. Thirty-three PAS proteins were discovered in PI3K inhibitor Xanthomonas campestris pv. campestris (Xcc) by genome/proteome analysis. Thirteen PAS proteins were identified as contributing to light signalling and Xcc growth, motility or virulence using molecular genetics and bioinformatics methods. The PAS domains played important roles in light signalling to regulate the growth, motility and virulence of Xcc. They might be regulated by not only light quality (wavelength) this website but also quantity (intensity) as potential light-signalling components. Evaluating the light wavelength, three light-signalling types of PAS proteins in Xcc were shown to be involved in blue light signalling, tricolour (blue, red and far-red) signalling or red/far-red signalling. This showed that Xcc had evolved a complicated

light-signalling system to adapt to a complex environment. Light is an energy source and an ever-present stimulus in the environment, and numerous studies have shown bacterial responses to light wavelengths spanning much of the visible spectrum (Konig et al., 2000; Bhoo et al., 2001; Lamparter et al., 2002; Pattison & Davies, 2006). The longer wavelengths, including red and far-red light, have been shown to regulate bacterial metabolism and a wide range of other responses (Bhoo et al., next 2001; Lamparter et al., 2002). Some responses to long-wavelength

light may be mediated by light-induced changes in protein structure. The vast majority of characterized bacteriophytochrome (Bph) proteins exhibit a reversible state of protein-red (Pr) to protein-far-red (Pfr) transition, which possibly corresponds to diverse, species-specific roles including phototaxis, chromatic adaptation, resetting circadian clocks and regulation of pigment biosynthesis. (Bhoo et al., 2001; Lamparter et al., 2002). The short-wavelength region, that is the ultraviolet (UV) and blue light ranges, can have powerful effects on organisms not only because of deleterious effects on DNA (UV range) (Pattison & Davies, 2006), but also the capability to excite, with high yield, ubiquitously present photosensitizing compounds, for example, porphyrins and flavins (Spikes, 1989). To date, most of our knowledge of putative light-sensing molecules in bacteria has come from bioinformatics prediction, with little experimental confirmation of their functions.

Another genomic fragment containing sciP and adjacent ctrA was amplified using primers sciP-comF and ctrA-comR for disrupting both genes. The KIXX cartridge replaced a 644-bp SmaI/BamHI fragment, deleting the last 215 bp of sciP and the first 331 bp of the 711 bp ctrA. Plasmids containing disrupted versions of the genes were conjugated to R. capsulatus from E. coli C600 (pDPT51) (Taylor et al., 1983). Mutant strains were generated by GTA transfer of the disrupted versions of the genes into the chromosome of SB1003 (Scolnik & Haselkorn, 1984). PCR

with the original amplification primers was Omipalisib mw used to confirm the resulting kanamycin-resistant strains contained only the disrupted genes. Plasmid recombineering (Noll et al., 2009) was used for the generation of the cckA deletion construct. Primers cckA-p1 and cckA-p2 (Table 1)

were used to amplify a 4351-bp region encoding cckA (rcc01749) plus ~1 kb of flanking Sirolimus clinical trial sequence on each side. Gel-purified PCR fragments were recombined into pUC19 (Vieira & Messing, 1982), and parental plasmids were selectively linearized by SalI treatment. Primers cckA-p3 and cckA-p4 (Table 1) were designed to PCR-amplify kanamycin resistance cassette A002 (Gene Bridges, Germany) with 50-bp tails homologous to cckA bases 53–103 and 2202–2252, respectively. λ-Red recombination resulted in the replacement of ~91% of plasmid-encoded cckA with the kanamycin resistance marker, yielding pUCΔcckA. The resulting plasmid was used to generate the cckA mutant strain as described above for the chpT, sciP and the ctrA/sciP double mutants.

Trans complementation of wild-type genes under control of their native upstream sequences was performed with the low copy number broad-host-range plasmid, pRK767 (Gill & Warren, 1988). The complementing fragments were amplified from the genome with appropriate primers (Table 1). Site-directed mutagenesis was performed with the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) to create pRK767-borne mutant ctrA genes with their native promoter region encoding a CtrA phosphomimetic protein, CtrAD51E (Domian et al., 1997), and a CtrA protein that is unable to be phosphorylated, CtrAD51A (Ryan et al., 2002), using primers D51E-F/D51E-R Etoposide mw and D51A-F/D51A-R, respectively (Table 1). The mutagenesis created single bp substitutions that resulted in a glutamate (D51E) or alanine (D51A) in place of the conserved aspartate (D51) phosphorylation site; the presence of the mutations was confirmed by sequencing. These plasmids and the empty pRK767 control were transferred to R. capsulatus via conjugation using E. coli S17-1 (Simon et al., 1983). RcGTA packages random fragments of the R. capsulatus genome and transfers these to recipient cells. A gene transfer bioassay was used to measure production and release of RcGTA particles.

Skilled motor practice facilitates the formation of an internal model of movement, which may then be used to anticipate task-specific requirements at a later time (Shadmehr & Holcomb, 1997). Internal models are most susceptible to interference during and immediately following practice and become less susceptible to interference

over time through persistent neural activity, a process called consolidation (Brashers-Krug et al., 1996; Robertson et al., 2004). Motor memory consolidation can take place both explicitly, with conscious awareness, or implicitly, without conscious awareness of the skill being performed (Robertson et al., 2004). The neural processes of consolidation can take two forms: (i) online improvements that occur KU-60019 datasheet concurrent with practice or (ii) offline improvements that develop following the termination

of practice (Brashers-Krug et al., 1996; Robertson et al., 2004). Importantly, these two processes are not completely independent or mutually exclusive. Given its known role in the selection of movements (Kalaska & Crammond, 1995; Rushworth et al., 2003) and implicit motor learning (Ohbayashi et al., 2003; Meehan et al., 2011), the dorsal premotor cortex (PMd) is a logical candidate for involvement in motor memory consolidation. Our group reported improved implicit sequence-specific motor learning when 5 Hz repetitive BEZ235 mw transcranial magnetic stimulation (rTMS) was delivered over the PMd prior to skilled motor practice of a continuous tracking task (Boyd & Linsdell, 2009). Yet it is not clear whether improvements noted when PMd stimulation precedes motor practice result from only online or a combination of online and offline consolidation

of sequence-specific elements. The current work sought to directly triclocarban assess the involvement of PMd in offline consolidation of skilled motor practice. In contrast to our previous work (Boyd & Linsdell, 2009), three groups received either 1 Hz, 5 Hz or control rTMS immediately following practice of a continuous visuomotor tracking task (Experiment 1). Based on our previous work, it was hypothesized that 5 Hz rTMS immediately following practice would enhance while 1 Hz rTMS would suppress motor learning compared with control stimulation. However, the effects of TMS are known to be ‘state dependent’ (Silvanto et al., 2008). State-dependence has been demonstrated during both perceptual and cognitive tasks where prior or concurrent neural activity (Silvanto et al., 2007b; Arai et al., 2011) and/or task-specific elements (Bestmann et al., 2008; Cohen & Robertson, 2011) influence expected outcomes. An alternative hypothesis is that 1 Hz rTMS, typically associated with inhibition, over PMd immediately following practice may enhance implicit sequence-specific motor learning through state-dependent mechanisms.

We investigated skeletal muscle form and function

by measuring force in response to both nerve-mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1-43 were impaired in mature NT-3-deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype-dependent variation in the Idelalisib price number of synaptic vesicles near the active zone. NT-3+/− mice had normal soleus muscle fiber numbers but their fibers had smaller cross-sectional areas and were more densely-packed than wild-type littermates. Moreover, the muscles of adult NT-3-deficient animals were weaker than those of wild-type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT-3 availability during development impairs motor nerve terminal maturation and synaptic vesicle

recycling and leads to a reduction in muscle fiber diameter. “
“Recent findings indicate that the hippocampus is not only crucial for long-term memory (LTM) encoding, but plays a role for working memory (WM) as well. In particular, it has been shown that the hippocampus is important for WM maintenance of multiple items or associations between item features. Previous studies Sirolimus supplier Anacetrapib using intracranial electroencephalography recordings from the hippocampus of patients with epilepsy revealed slow positive potentials during maintenance of a single item and during LTM encoding, but slow negative potentials during maintenance

of multiple items. These findings predict that WM maintenance of multiple items interferes with LTM encoding, because these two processes are associated with slow potentials of opposing polarities in the hippocampus. Here, we tested this idea in a dual-task paradigm involving a LTM encoding task nested into a WM Sternberg task with either a low (one item) or a high (three items) memory load. In the high WM load condition, LTM encoding was significantly impoverished, and slow hippocampal potentials were more negative than in the low WM load condition. Time-frequency analysis revealed that a reduction of slow hippocampal activity in the delta frequency range supported LTM formation in the low load condition, but not during high WM load. Together, these findings indicate that multi-item WM and LTM encoding interfere within the hippocampus. “
“The rodent ventrobasal (VB) thalamus receives sensory inputs from the whiskers and projects to the cortex, from which it receives reciprocal excitatory afferents.